Abstract
BackgroundPlasmodium falciparum, the deadliest causative agent of malaria, has high prevalence in Nigeria. Drug resistance causing failure of previously effective drugs has compromised anti-malarial treatment. On this basis, there is need for a proactive surveillance for resistance markers to the currently recommended artemisinin-based combination therapy (ACT), for early detection of resistance before it become widespread.MethodsThis study assessed anti-malarial resistance genes polymorphism in patients with uncomplicated P. falciparum malaria in Lagos, Nigeria. Sanger and Next Generation Sequencing (NGS) methods were used to screen for mutations in thirty-seven malaria positive blood samples targeting the P. falciparum chloroquine-resistance transporter (Pfcrt), P. falciparum multidrug-resistance 1 (Pfmdr1), and P. falciparum kelch 13 (Pfk13) genes, which have been previously associated with anti-malarial resistance.ResultsExpectedly, the NGS method was more proficient, detecting six Pfmdr1, seven Pfcrt and three Pfk13 mutations in the studied clinical isolates from Nigeria, a malaria endemic area. These mutations included rare Pfmdr1 mutations, N504K, N649D, F938Y and S967N, which were previously unreported. In addition, there was moderate prevalence of the K76T mutation (34.6%) associated with chloroquine and amodiaquine resistance, and high prevalence of the N86 wild type allele (92.3%) associated with lumefantrine resistance.ConclusionWidespread circulation of mutations associated with resistance to current anti-malarial drugs could potentially limit effective malaria therapy in endemic populations.
Highlights
Plasmodium falciparum, the deadliest causative agent of malaria, has high prevalence in Nigeria
Detection of P. falciparum kelch 13 (Pfk13), Pfcrt, andPfmdr1 mutations by Sanger and Next Generation Sequencing (NGS) methods Clinical samples from 1500 patients in Ikorodu and 730 in Amukoko study sites which were screened for malaria by rapid diagnostic test (RDT) and microscopy yielded 98 malaria-positive samples that were evaluated further by photo-induced electron transfer (PET)-PCR methods
In order to determine the efficiency of using the Sanger and NGS methods to detect mutations in the clinical isolates, both methods were used for sequencing of the selected genes
Summary
Plasmodium falciparum, the deadliest causative agent of malaria, has high prevalence in Nigeria. Drug resistance causing failure of previously effective drugs has compromised anti-malarial treatment On this basis, there is need for a proactive surveillance for resistance markers to the currently recommended artemisinin-based combination therapy (ACT), for early detection of resistance before it become widespread. Deployment of ACT has led to a significant reduction in the number of malaria cases and fatalities [3], resistance to artemisinin, manifesting as delayed parasite clearance, has been observed in Western Cambodia since 2008 and has more recently been documented in extended areas of Southeast Asia [6,7,8,9]. Mutations in the kelch propeller domain of the k13 gene have been identified as molecular markers for artemisinin resistance by evaluating in vitro survival assays and observing delayed parasite clearance times and are currently being used to track the spread of resistance [7]. The substitution at codon 76 (K76T) is the most predictive point mutation for chloroquine resistance [13]
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